Rabbit anti cre

Pancreata were dissected, fixed, and processed as previously described (Golson et al. 2010). Antibodies used included guinea pig anti‐insulin (1:500; Dako, Carpinteria, CA), rabbit anti‐Ki67 (1:500; AbCam, Cambridge, MA), rabbit anti‐Cre (1:2000, Millipore, San Diego, CA), Cy2‐conjugated anti‐guinea pig IgG (1:400; Jackson ImmunoResearch Laboratories, West Grove, PA), Cy3‐conjugated anti‐rabbit IgG (1:400; Jackson ImmunoResearch Laboratories, West Grove, PA), and horseradish peroxidase‐conjugated anti‐guinea pig IgG (1:400; Jackson ImmunoResearch Laboratories, West Grove, PA). Nuclei were visualized with 4′,6′‐diamidino‐2‐phenylindole (DAPI, 1 μg/mL; Molecular Probes, Grand Island, NY). The antigen retrieval used for Ki67 immunolabeling consisted of microwaving slides for 14 min on high power in 10 mmol/L sodium citrate buffer pH 6.0. The antigen retrieval required for Cre immunostaining consisted of microwaving slides on high for 1 minute followed by 7.5 min at 10% power in 1× Tris‐EGTA buffer (TEG) pH 9.0.

Immunohistochemistry was performed as previously described [26 ]. Briefly, postnatal and embryonic brains were collected and placed in 4% paraformaldehyde overnight at 4 °C, cryoprotected in 30% sucrose for at least 24 h, frozen in optimal cutting temperature and cryosectioned. All tissues were sectioned coronally at 12 or 20 μm and stained on glass slides.
For SP9, BCL11B, and SIX3 immunohistochemistry, sections were boiled briefly in 10 mmol/L sodium citrate for antigen retrieval. Immunohistochemistry for BrdU⁺ cells was performed after 45 min of incubation in 2 N HCl and rinsing twice in 0.1 mol/L borate buffer at room temperature. Immunofluorescence labeling was performed with the following primary antibodies: rat anti-BCL11B (Abcam, Ab18465), rat anti-BrdU (Accurate Chemical, OBT0030s), chicken anti-β-gal (Abcam, ab9361), rabbit anti-cleaved Caspase-3 (Cell Signaling, #9661), rabbit anti-CRE (Millipore, 69050-3), rabbit anti-EBF1 (Merck, AB10523), rabbit anti-FOXP1 (Abcam, Ab16645), rabbit anti-KI67 (Abcam, ab15580), mouse anti-SIX3 (Santa Cruz Biotechnology, sc-398797), goat anti-SP8 (Santa Cruz Biotechnology, sc-104661), rabbit anti-SP9 [25 (link)]. Appropriate Alexa Fluor 488-, Cy3- or Alexa Fluor 647-conjugated secondary antibodies from Jackson ImmunoResearch were used.